Mass spectrometric determination of fatty acids

ABSTRACT

The invention relates to the detection of fatty acids. In a particular aspect, the invention relates to methods for detecting very long chain fatty acids and branched chain fatty acids by mass spectrometry.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/233,322, filed Aug. 10, 2016, now U.S. Pat. No. 10,242,852, which isa continuation of U.S. application Ser. No. 14/242,075, filed Apr. 1,2014, now U.S. Pat. No. 9,449,801, which is a continuation of U.S.application Ser. No. 13/529,844, filed Jun. 21, 2012, now U.S. Pat. No.8,728,824, which claims priority to U.S. Provisional Patent ApplicationNo. 61/500,002, filed Jun. 22, 2011. The entire contents of theforegoing applications are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The invention relates to the quantitative measurement of fatty acids. Ina particular aspect, the invention relates to methods for quantitativemeasurement of fatty acids by APCI-mass spectrometry.

BACKGROUND OF THE INVENTION

Peroxisomes of eukaryotic cells break down fatty acids that are too longto be oxidized by mitochondria. Two types of fatty acids that are brokendown in peroxisomes are very long chain fatty acids (VLCFA) and branchedchain fatty acid (BCFA). VLCFA are a family of saturated fatty acidsthat have branched or unbranched aliphatic tails of 22 carbons or more.Examples of VLCFA with unbranched aliphatic tails include docosanoicacid, tetracosanoic acid, and hexacosanoic acid. Docosanoic acid (alsoknown as behenic acid) is an unbranched saturated fatty acid having a 22carbon chain. Tetracosanoic acid (also known as lignoceric acid) is anunbranched saturated fatty acid having a 24 carbon chain. Hexacosanoicacid (also known as cerotic acid) is an unbranched saturated fatty acidhaving a 26 carbon chain. BCFA are saturated or unsaturated fatty acidswith aliphatic tails less than 22 carbons long in which other chemicalgroups, such as methyl groups, extend (i.e., branch) from the aliphatictail. One example of a BCFA is phytanic acid. Phytanic acid (also knownas 3,7,11,15-tetramethyl hexadecanoic acid) is a saturated BCFA 16carbons long with methyl groups on the 3^(rd), 7^(th), 11^(th), and15^(th) carbons from the carboxylic end of the molecule. Another exampleof a BCFA is pristanic acid (also known as2,6,10,14-tetramethylpentadecanoic acid) is a saturated BCFA 15 carbonslong with methyl groups on the 2^(nd), 6^(th), 10^(th), and 14^(th)carbons from the carboxylic end of the molecule.

Peroxisomes break down VLCFA by β-oxidation. Some BCFA, such as phytanicacid, cannot undergo β-oxidation due to their particular branchedstructure and are initially broken down in peroxisomes by α-oxidation.For example, phytanic acid is broken down into pristanic acid throughα-oxidation, and the pristanic acid is then able to undergo furtherbreakdown via β-oxidation.

Peroxisomal disorders are generally characterized by the inability ofperoxisomes to break down VLCFA and BCFA. These disorders include, butare not limited to, Zellweger syndrome, pseudo-Zellweger syndrome,infantile and adult Refsum disease, adrenoleukodystrophy, rhizomelicchondrodysplasia punctata type 1 (RCDP-1), D-bifunctional proteindeficiency, and acyl-coA oxidase deficiency. Patients suffering fromthese types of disorders can accumulate VLCFA and BCFA in their bloodand tissue because peroxisomes in these disorders are unable toadequately breakdown VLCFA and BCFA. Thus, it is desirable to be able todetect levels of VLCFA and/or BCFA or their breakdown products in asubject to aid in diagnosis of these disorders.

Quantitation of certain VLCFA by liquid chromatography-mass spectrometry(LC-MS) has been reported. For example, Butovich reports quantitation ofderivatized docosanoic acid, tetracosanoic acid, and hexacosanoic acidin meibum by HPLC-APCI (positive ion)-MS (J. Lipid Res. 2009. 50:501-513); Lam et al. reports quantitation of fatty acids such as oleicacid, linoleic acid, and linolenic acid by LC-ESI (negative ion)-MS fromin vitro enzymatic cleavage of plant oils (U.S. Pub. No. 2008/0305531).Al-Dirbashi et al. report quantitation of derivatized VLCFA and BCFAfrom plasma, using LC-ESI (positive ion)-MS/MS (J. Lipid Res. 2008. 49:1855-1862).

Other mass spectrometric methods have been reported for quantitation ofBCFA. For example, Verhoeven et al. describe using GC-NCI-MS as well asGC-MS/MS to quantitate derivatized BCFA from plasma and culturedfibroblasts, respectively (see J. Lipid Res. 1999. 40:260-266; and J.Lipid Res. 1998. 39:66-74). Fernandusse et al. report using GC-MS toquantitate derivatized BCFA from plasma (J. Lipid Res. 2002. 43:438-444). In addition, ten Brink et al. report using GC-MS and electroncapture NCI to quantitate derivatized BCFA from plasma (see J. LipidRes. 1992. 33: 1449-1457; and J. Lipid Res. 1992. 33: 41-47).

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the amount of fattyacids in a sample by mass spectrometry.

In one aspect, the invention provides methods for determining the amountof one or more very long chain fatty acids (VLCFA) and/or branched chainfatty acids (BCFA) in a sample by mass spectrometry. In someembodiments, the method comprises the steps of: (a) subjecting one ormore VLCFA and/or BCFA from the sample to an atmospheric pressurechemical ionization (APCI) source to generate one or more VLCFA and/orBCFA ions detectable by mass spectrometry; (b) determining the amount ofone or more of the VLCFA and/or BCFA ions by mass spectrometry; and (c)relating the amount of the VLCFA and/or BCFA ions to the amount of theVLCFA and/or BCFA in the sample.

In another aspect, the invention provides a method of determining theamount of one or more very long chain fatty acids (VLCFA) and/orbranched chain fatty acids (BCFA) in a plasma or serum sample from ahuman patient comprising: (a) subjecting the sample to a hydrolyzingagent to generate a hydrolyzed sample; (b) purifying the hydrolyzedsample to generate a purified sample; (c) subjecting the purified sampleto an atmospheric pressure chemical ionization (APCI) source to generateone or more VLCFA and/or BCFA ions detectable by mass spectrometry; (d)determining the amount of one or more VLCFA and/or BCFA ions by massspectrometry; and (e) relating the amount of VLCFA and/or BCFA ionsdetermined in step (d) to the amount of the VLCFA and/or BCFA in thesample.

In some embodiments, the amounts of one or more VLCFA are determined. Insome of these embodiments, the one or more VLCFA are selected from thegroup consisting of docosanoic acid, tetracosanoic acid, andhexacosanoic acid. In embodiments where the VLCFA comprises docosanoicacid, the VLCFA ions may comprise ions with a mass to charge ratio (m/z)of 339.3±0.5. In embodiments where the VLCFA comprises tetracosanoicacid, the VLCFA ions may comprise ions with a mass to charge ratio (m/z)of 367.3±0.5. In embodiments where the VLCFA comprises hexacosanoicacid, the VLCFA ions may comprise ions with a mass to charge ratio (m/z)of 395.4±0.5.

In some embodiments, the amounts of one or more BCFA are determined. Insome of these embodiments, the BCFA are methyl-BCFA, such as a BCFAselected from the group consisting of pristanic acid and phytanic acid.In embodiments where the BCFA comprises pristanic acid, the BCFA ionsmay comprise ions with a mass to charge ratio (m/z) of 297.3±0.5. Inembodiments where the BCFA comprises phytanic acid, the BCFA ions maycomprise ions with a mass to charge ratio (m/z) of 311.2±0.5.

In some embodiments, the APCI source is operating in negative ionizationmode. In some embodiments, the amounts of one or more VLCFA and one ormore BCFA are determined. In some embodiments, the VLCFA are selectedfrom the group consisting of docosanoic acid, tetracosanoic acid, andhexacosanoic acid. In some embodiments, the BCFA are selected from thegroup consisting of pristanic acid and phytanic acid. In someembodiments, the fatty acid analytes are underivatized prior toionization. In some embodiments, the sample comprises a biologicalsample, such as a sample derived from a human, such as plasma or serum.In some embodiments, the sample is subjected to a hydrolyzing agentprior to ionization. In some embodiments, the hydrolyzing agent is anacid or a base. In some embodiments, the sample is subjected toliquid/liquid extraction prior to ionization. In some embodiments, theVLCFA and/or BCFA are subjected to a liquid chromatography column priorto ionization. In some embodiments, the liquid chromatography columncomprises a high performance liquid chromatography (HPLC) column.

In some embodiments, the human patient is suspected of having aperoxisomal disorder, such as Zellweger syndrome, pseudo-Zellwegersyndrome, infantile and adult Refsum disease, adrenoleukodystrophy,rhizomelic chondrodysplasia punctata type 1 (RCDP-1), D-bifunctionalprotein deficiency, and acyl-coA oxidase deficiency. In someembodiments, VLCFA and/or BCFA are quantitated in a patient sample todiagnose, prognose, or monitor the treatment of a patient with aperoxisomal disorder.

As used herein, “very long chain fatty acids” (or “VLCFA”) are fattyacids with aliphatic tails of 22 carbons or longer in length. Thealiphatic tails of VLCFA may be branched or unbranched, and saturated orunsaturated. In certain embodiments, the methods described herein may beused to determine the amount of one or more unbranched saturated VLCFAin a sample. Examples of unbranched, saturated VLCFA include docosanoicacid, tetracosanoic acid, hexacosanoic acid.

As used herein, “branched chain fatty acids” (or “BCFA”) are fatty acidswith aliphatic tails less than 22 carbons long that have side chains (or“branches”). Typically, the side chains are methyl groups, and the sidechains may occur at one or more locations along the aliphatic tail. Incertain embodiments, the methods described herein are used to determinethe amount of one or more methyl-branched chain fatty acids(methyl-BCFA) in a sample. Examples of methyl-BCFA include pristanicacid and phytanic acid.

As used herein, “derivatizing” means reacting two or more molecules toform a new molecule. As used herein, the names of derivatized forms ofcompounds (including fatty acids such as phytanic acid and docosanoicacid) include an indication as to the nature of derivatization. Forexample, the methyl esters of phytanic acid and docosanoic acid would bereferred to as phytanic acid-methyl ester and docosanoic acid-methylester.

Mass spectrometry may be performed in negative ion mode. Alternatively,mass spectrometry may be performed in positive ion mode. Variousionization sources, including for example atmospheric pressure chemicalionization (APCI), laser diode thermal desorption (LDTD), orelectrospray ionization (ESI), may be used in embodiments of the presentinvention. In certain preferred embodiments, VLCFA and/or BCFA aremeasured using APCI in negative ion mode.

In preferred embodiments, one or more separately detectable internalstandards are provided in the sample, the amount of which are alsodetermined in the sample. In these embodiments, all or a portion of boththe analyte(s) of interest and the internal standard(s) present in thesample are ionized to produce a plurality of ions detectable in a massspectrometer, and one or more ions produced from each analyte ofinterest and internal standard are detected by mass spectrometry.Exemplary internal standards for fatty acids include pristanic acid-²H₃phytanic acid-²H₃, docosanoic acid-²H₃, tetracosanoic acid-²H₃, andhexacosanoic acid-²H₃.

Ions detectable in a mass spectrometer may be generated for each of theexemplary internal standards listed above. Exemplary spectra generateddemonstrating detection of pristanic acid-²H₃, phytanic acid-²H₃,docosanoic acid-²H₃, tetracosanoic acid-²H₃, and hexacosanoic acid-²H₃are discussed in Example 4, and shown in FIGS. 6-10, respectively.

As used herein, an “isotopic label” produces a mass shift in the labeledmolecule relative to the unlabeled molecule when analyzed by massspectrometric techniques. Examples of suitable labels include deuterium(d or ²H), ¹³C, and ¹⁵N. For example, phytanic acid-²H₃ and docosanoicacid-²H₃ have masses of about 3 mass units higher than unlabeledphytanic acid and docosanoic acid. The isotopic label can beincorporated at one or more positions in the molecule and one or morekinds of isotopic labels can be used on the same isotopically labeledmolecule.

In other embodiments, amounts of VLCFA and/or BCFA ions may bedetermined by comparison to one or more external reference standards.Exemplary external reference standards include blank plasma or serumspiked with one or more of pristanic acid-²H₃, phytanic acid-²H₃,docosanoic acid-²H₃, tetracosanoic acid-²H₃, and hexacosanoic acid-²H₃.External standards typically will undergo the same treatment andanalysis as any other sample to be analyzed.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedparent or daughter ions by mass spectrometry. Relative reduction as thisterm is used does not require that any substance, present with theanalyte of interest in the material to be purified, is entirely removedby purification.

As used herein, the term “solid phase extraction” or “SPE” refers to aprocess in which a chemical mixture is separated into components as aresult of the affinity of components dissolved or suspended in asolution (i.e., mobile phase) for a solid through or around which thesolution is passed (i.e., solid phase). In some instances, as the mobilephase passes through or around the solid phase, undesired components ofthe mobile phase may be retained by the solid phase resulting in apurification of the analyte in the mobile phase. In other instances, theanalyte may be retained by the solid phase, allowing undesiredcomponents of the mobile phase to pass through or around the solidphase. In these instances, a second mobile phase is then used to elutethe retained analyte off of the solid phase for further processing oranalysis. SPE, including TFLC, may operate via a unitary or mixed modemechanism. Mixed mode mechanisms utilize ion exchange and hydrophobicretention in the same column; for example, the solid phase of amixed-mode SPE column may exhibit strong anion exchange and hydrophobicretention; or may exhibit strong cation exchange and hydrophobicretention.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and turbulent flow liquidchromatography (TFLC) (sometimes known as high turbulence liquidchromatography (HTLC) or high throughput liquid chromatography).

As used herein, the term “high performance liquid chromatography” or“HPLC” (sometimes known as “high pressure liquid chromatography”) refersto liquid chromatography in which the degree of separation is increasedby forcing the mobile phase under pressure through a stationary phase,typically a densely packed column.

As used herein, the term “turbulent flow liquid chromatography” or“TFLC” (sometimes known as high turbulence liquid chromatography or highthroughput liquid chromatography) refers to a form of chromatographythat utilizes turbulent flow of the material being assayed through thecolumn packing as the basis for performing the separation. TFLC has beenapplied in the preparation of samples containing two unnamed drugs priorto analysis by mass spectrometry. See, e.g., Zimmer et al., JChromatogr. A 854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367,5,919,368, 5,795,469, and 5,772,874, which further explain TFLC. Personsof ordinary skill in the art understand “turbulent flow”. When fluidflows slowly and smoothly, the flow is called “laminar flow”. Forexample, fluid moving through an HPLC column at low flow rates islaminar. In laminar flow the motion of the particles of fluid is orderlywith particles moving generally in straight lines. At faster velocities,the inertia of the water overcomes fluid frictional forces and turbulentflow results. Fluid not in contact with the irregular boundary “outruns”that which is slowed by friction or deflected by an uneven surface. Whena fluid is flowing turbulently, it flows in eddies and whirls (orvortices), with more “drag” than when the flow is laminar. Manyreferences are available for assisting in determining when fluid flow islaminar or turbulent (e.g., Turbulent Flow Analysis: Measurement andPrediction, P. S. Bernard & J. M. Wallace, John Wiley & Sons, Inc.,(2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott,Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 50 μm.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. In a preferredembodiment, the analytical column contains particles of about 5 μm indiameter. Such columns are often distinguished from “extractioncolumns”, which have the general purpose of separating or extractingretained material from non-retained materials in order to obtain apurified sample for further analysis.

As used herein, the terms “on-line” and “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged species; and (2) detecting thecharged species based on their mass-to-charge ratio. The compounds maybe ionized and detected by any suitable means. A “mass spectrometer”generally includes an ionizer and an ion detector. In general, one ormore molecules of interest are ionized, and the ions are subsequentlyintroduced into a mass spectrometric instrument where, due to acombination of magnetic and electric fields, the ions follow a path inspace that is dependent upon mass (“m”) and charge (“z”). See, e.g.,U.S. Pat. No. 6,204,500, entitled “Mass Spectrometry From Surfaces;”U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem MassSpectrometry;” U.S. Pat. No. 6,268,144, entitled “DNA Diagnostics BasedOn Mass Spectrometry;” U.S. Pat. No. 6,124,137, entitled“Surface-Enhanced Photolabile Attachment And Release For Desorption AndDetection Of Analytes;” Wright et al., Prostate Cancer and ProstaticDiseases 1999, 2: 264-76; and Merchant and Weinberger, Electrophoresis2000, 21: 1164-67.

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber, which is heated slightly to prevent condensation and toevaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to a form of ionization where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Anal. Chem.2000, 72 (15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase. Laser diode thermal desorption (LDTD) is a technique wherein asample containing the analyte is thermally desorbed into the gas phaseby a laser pulse. The laser hits the back of a specially made 96-wellplate with a metal base. The laser pulse heats the base and the heatcauses the sample to transfer into the gas phase. The gas phase samplemay then be drawn into an ionization source, where the gas phase sampleis ionized in preparation for analysis in the mass spectrometer. Whenusing LDTD, ionization of the gas phase sample may be accomplished byany suitable technique known in the art, such as by ionization with acorona discharge (for example by APCI).

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, the term “lower limit of quantification”, “lower limitof quantitation” or “LLOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LLOQ isidentifiable, discrete and reproducible with a concentration at whichthe standard deviation (SD) is less than one third of the totalallowable error (TEa).

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as the mean of the blankplus four times the standard deviation of the blank.

As used herein, an “amount” of an analyte in a body fluid sample refersgenerally to an absolute value reflecting the mass of the analytedetectable in volume of sample. However, an amount also contemplates arelative amount in comparison to another analyte amount. For example, anamount of an analyte in a sample can be an amount which is greater thana control or normal level of the analyte normally present in the sample.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show exemplary chromatograms for pristanic acid (FIG.1A) and pristanic acid-²H₃ (internal standard; FIG. 1B). Details arediscussed in Example 4.

FIGS. 2A and 2B show exemplary chromatograms for phytanic acid (FIG. 2A)and phytanic acid-²H₃ (internal standard; FIG. 2B). Details arediscussed in Example 4.

FIGS. 3A and 3B show exemplary chromatograms for docosanoic acid (FIG.3A) and docosanoic acid-²H₃ (internal standard; FIG. 3B). Details arediscussed in Example 4.

FIGS. 4A and 4B show exemplary chromatograms for tetracosanoic acid(FIG. 4A) and tetracosanoic acid-²H₃ (internal standard; FIG. 4B).Details are discussed in Example 4.

FIGS. 5A and 5B show exemplary chromatograms for hexacosanoic acid (FIG.5A) and hexacosanoic acid-²H₃ (internal standard; FIG. 5B). Details arediscussed in Example 4.

FIGS. 6A and 6B show an exemplary chromatogram (FIG. 6A) and spectrum(FIG. 6B) demonstrating detection of pristanic acid and pristanicacid-²H₃ (internal standard) in a serum sample. Details are discussed inExample 4.

FIGS. 7A and 7B show an exemplary chromatogram (FIG. 7A) and spectrum(FIG. 7B) demonstrating detection of phytanic acid/phytanic acid-²H₃(internal standard) in a serum sample. Details are discussed in Example4.

FIGS. 8A and 8B show an exemplary chromatogram (FIG. 8A) and spectrum(FIG. 8B) demonstrating detection of docosanoic acid/docosanoic acid-²H₃(internal standard) in a serum sample. Details are discussed in Example4.

FIGS. 9A and 9B show an exemplary chromatogram (FIG. 9A) and spectra(FIG. 9B) demonstrating detection of tetracosanoic acid/tetracosanoicacid-²H₃ (internal standard) in a serum sample. Details are discussed inExample 4.

FIGS. 10A and 10B show an exemplary chromatogram (FIG. 10A) and spectrum(FIG. 10B) demonstrating detection of hexacosanoic acid/hexacosanoicacid-²H₃ (internal standard) in a serum sample. Details are discussed inExample 4.

FIG. 11 shows an exemplary calibration curve generated for pristanicacid. Details are discussed in Example 4.

FIG. 12 shows an exemplary calibration curve generated for phytanicacid. Details are discussed in Example 4.

FIG. 13 shows an exemplary calibration curve generated for docosanoicacid. Details are discussed in Example 4.

FIG. 14 shows an exemplary calibration curve generated for tetracosanoicacid. Details are discussed in Example 4.

FIG. 15 shows an exemplary calibration curve generated for hexacosanoicacid. Details are discussed in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for measuring fatty acids in a sample. Morespecifically, mass spectrometric methods are described for detecting andquantifying fatty acids in a sample. The methods may utilize APCI toionize underivatized VLCFA and/or BCFA in the sample prior to detectionby mass spectrometry.

The methods may use an on-line analytical liquid chromatographytechnique, such as high performance liquid chromatography (HPLC), toperform a purification of VLCFA and/or BCFA, combined with methods ofmass spectrometry (MS), thereby providing a high-throughput assay systemfor detecting and quantifying fatty acids in a sample. Preferredembodiments are particularly well suited for application in largeclinical laboratories for automated VLCFA and/or BCFA quantitation.

Suitable test samples for use in methods of the present inventioninclude any test sample that may contain the analyte of interest. Insome preferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Preferred samples comprise bodilyfluids such as blood, plasma, serum, saliva, cerebrospinal fluid, ortissue samples; preferably plasma (including EDTA and heparin plasma)and serum. Such samples may be obtained, for example, from a patient;that is, a living person, male or female, presenting oneself in aclinical setting for diagnosis, prognosis, or treatment of a disease orcondition.

The present invention also contemplates kits for a VLCFA and/or BCFAquantitation assay. A kit for a VLCFA and/or BCFA quantitation assay mayinclude a kit comprising the compositions provided herein. For example,a kit may include packaging material and measured amounts of packagedreagents, including an isotopically labeled internal standard, inamounts sufficient for at least one assay. Typically, the kits will alsoinclude instructions recorded in a tangible form (e.g., contained onpaper or an electronic medium) for using the packaged reagents for usein a VLCFA and/or BCFA quantitation assay.

Calibration and QC pools for use in embodiments of the present inventionare preferably prepared using a matrix similar to the intended samplematrix.

Sample Preparation for Mass Spectrometric Analysis

In preparation for mass spectrometric analysis, VLCFA and/or BCFA(including pristanic acid, phytanic acid, docosanoic acid, tetracosanoicacid, and/or hexacosanoic acid) in the sample may be enriched relativeto their ester counterparts by hydrolysis of fatty acid esters by anytechnique known in the art. In some embodiments, fatty acid esters inthe sample are hydrolyzed by contacting the sample with a strong acid(e.g., HCl) or a strong base (e.g., NaOH) and optionally incubating atan elevated temperature, such as about 120° C. to about 125° C. Theincubation period may vary depending on the amount of sample andconcentration of acid used. Certain embodiments described herein utilizean incubation period of about 60 minutes to hydrolyze 200 μL of sample,diluted with 100 μL of internal standard, with 200 μL of 1 M NaOH. Afterincubation, excess hydroxide may be neutralized by treatment with anacid, such as hydrochloric acid (HCl).

Additionally, VLCFA and/or BCFA may be enriched relative to one or moreother components in the sample (e.g. protein) by various methods knownin the art, including for example any combination of liquidchromatography, filtration, centrifugation, thin layer chromatography(TLC), electrophoresis including capillary electrophoresis, affinityseparations including immunoaffinity separations, extraction methodsincluding ethyl acetate or methanol extraction, and the use ofchaotropic agents or any combination of the above or the like. If bothhydrolysis and purification steps are used, purification is preferablyconducted after hydrolysis.

Protein precipitation is one method of preparing a test sample,especially a biological test sample, such as serum or plasma. Proteinpurification methods are well known in the art, for example, Polson etal., Journal of Chromatography B 2003, 785:263-275, describes proteinprecipitation techniques suitable for use in methods of the presentinvention. Protein precipitation may be used to remove most of theprotein from the sample leaving fatty acids in the supernatant. Thesamples may be centrifuged to separate the liquid supernatant from theprecipitated proteins; alternatively the samples may be filtered toremove precipitated proteins. The resultant supernatant or filtrate maythen be applied directly to mass spectrometry analysis; or alternativelyto liquid chromatography and subsequent mass spectrometry analysis. Incertain embodiments, samples, such as plasma or serum, may be purifiedby a hybrid protein precipitation/liquid-liquid extraction method. Inthese embodiments, a sample is mixed with methanol, ethyl acetate, andwater, and the resulting mixture is vortexed and centrifuged. Theresulting supernatant is removed, dried to completion and reconstitutedin a suitable solvent. In certain embodiments described herein, thesolvent used to reconstitute the dried supernatant is ethanol.

Another method of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Certain methods of liquidchromatography, including HPLC, rely on relatively slow, laminar flowtechnology. Traditional HPLC analysis relies on column packing in whichlaminar flow of the sample through the column is the basis forseparation of the analyte of interest from the sample. The skilledartisan will understand that separation in such columns is a diffusionalprocess and may select LC, including HPLC, instruments and columns thatare suitable for use with fatty acids. The chromatographic columntypically includes a medium (i.e., a packing material) to facilitateseparation of chemical moieties (i.e., fractionation). The medium mayinclude minute particles, or may include a monolithic material withporous channels. A surface of the medium typically includes a bondedsurface that interacts with the various chemical moieties to facilitateseparation of the chemical moieties. One suitable bonded surface is ahydrophobic bonded surface such as an alkyl bonded, cyano bondedsurface, or highly pure silica surface. Alkyl bonded surfaces mayinclude C-4, C-8, C-12, or C-18 bonded alkyl groups. In preferredembodiments, the column is a C-18 alkyl bonded column (such as a BDSHYPERSIL™ (C18 alkyl chain) column from Thermo Scientific). Thechromatographic column includes an inlet port for receiving a sample andan outlet port for discharging an effluent that includes thefractionated sample. The sample may be supplied to the inlet portdirectly, or from an extraction column, such as an on-line SPE cartridgeor a TFLC extraction column.

In one embodiment, the sample may be applied to the LC column at theinlet port, eluted with a solvent or solvent mixture, and discharged atthe outlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytypic (i.e.mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition may be employed where the analyte of interest is retained bythe column, and a second mobile phase condition may subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, HPLC is conducted with an alkyl bondedanalytical column chromatographic system. In certain preferredembodiments, a C-18 alkyl bonded column (such as a BDS HYPERSIL™ (C18alkyl chain) column from Thermo Scientific) is used. In certainembodiments, HPLC is performed using 20 mM ammonium acetate as mobilephase A and 100% acetonitrile as mobile phase B.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, an extraction column may be used for purificationof VLCFA and/or BCFA prior to mass spectrometry. In such embodiments,samples may be extracted using an extraction column which captures theanalyte, then eluted and chromatographed on a second extraction columnor on an analytical HPLC column prior to ionization. For example, sampleextraction with a TFLC extraction column may be accomplished with alarge particle size (e.g. greater than 50 μm) packed column. Sampleeluted off of this column may then be transferred to an HPLC analyticalcolumn for further purification prior to mass spectrometry. Because thesteps involved in these chromatography procedures may be linked in anautomated fashion, the requirement for operator involvement during thepurification of the analyte can be minimized. This feature may result insavings of time and costs, and eliminate the opportunity for operatorerror.

In some embodiments, purification of VLCFA and/or BCFA is accomplishedwith liquid-liquid extraction. Liquid/liquid extraction may beaccomplished by adding a suitable quantity of an organic solvent, suchas 10% ethyl acetate in hexane, to the sample. This mixture is thenagitated, such as by vortexing, and chilled, and the organic layer isdecanted off for further analysis. In some embodiments, VLCFA and/orBCFA in the sample may be purified by liquid/liquid extraction followedby liquid chromatography prior to mass spectrometric analysis.

Detection and Quantitation by Mass Spectrometry

Mass spectrometry is performed using a mass spectrometer, which includesan ionization source for ionizing the fractionated sample and creatingcharged molecules for further analysis. For example, ionization of thesample may be performed by electron ionization, chemical ionization,electrospray ionization (ESI), photon ionization, atmospheric pressurechemical ionization (APCI), photoionization, atmospheric pressurephotoionization (APPI), laser diode thermal desorption (LDTD), fast atombombardment (FAB), liquid secondary ionization (LSI), matrix assistedlaser desorption ionization (MALDI), field ionization, field desorption,thermospray/plasmaspray ionization, surface enhanced laser desorptionionization (SELDI), inductively coupled plasma (ICP) and particle beamionization. In preferred embodiments, VLCFA and/or BCFA in the sampleare ionized by APCI.

Mass spectrometric techniques may be conducted in positive or negativeionization mode. In preferred embodiments, VLCFA and/or BCFA are ionizedin negative ionization mode.

In mass spectrometry techniques generally, after the sample has beenionized, the positively or negatively charged ions created thereby maybe analyzed to determine a mass-to-charge ratio. Suitable analyzers fordetermining mass-to-charge ratios include quadrupole analyzers, iontraps analyzers, and time-of-flight analyzers. Exemplary ion trapmethods are described in Bartolucci, et al., Rapid Commun. MassSpectrom. 2000, 14:967-73.

Ions in a MS system may be detected using several detection modes. Forexample, selected ions may be detected, i.e. using a selective ionmonitoring mode (SIM), or alternatively, mass transitions resulting fromcollision induced dissociation or neutral loss may be monitored, e.g.,multiple reaction monitoring (MRM) or selected reaction monitoring(SRM). Preferably, the mass-to-charge ratio is determined using aquadrupole analyzer. For example, in a “quadrupole” or “quadrupole iontrap” instrument, ions in an oscillating radio frequency fieldexperience a force proportional to the DC potential applied betweenelectrodes, the amplitude of the RF signal, and the mass/charge ratio.The voltage and amplitude may be selected so that only ions having aparticular mass/charge ratio travel the length of the quadrupole, whileall other ions are deflected. Thus, quadrupole instruments may act asboth a “mass filter” and as a “mass detector” for the ions injected intothe instrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

Alternate modes of operating a tandem mass spectrometric instrumentinclude product ion scanning and precursor ion scanning. For adescription of these modes of operation, see, e.g., E. Michael Thurman,et al., Chromatographic-Mass Spectrometric Food Analysis for TraceDetermination of Pesticide Residues, Chapter 8 (Amadeo R.Fernandez-Alba, ed., Elsevier 2005) (387).

The results of an analyte assay may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, external standards may be run with thesamples, and a standard curve constructed based on ions generated fromthose standards. Using such a standard curve, the relative abundance ofa given ion may be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of VLCFAand/or BCFA in the sample. Methods of generating and using such standardcurves are well known in the art and one of ordinary skill is capable ofselecting an appropriate internal standard. For example, in someembodiments one or more isotopically labeled fatty acids (e.g.,pristanic acid-²H₃, phytanic acid-²H₃, docosanoic acid-²H₃,tetracosanoic acid-²H₃, hexadocosanoic acid-²H₃) may be used as internalstandards. Numerous other methods for relating the amount of an ion tothe amount of the original molecule will be well known to those ofordinary skill in the art.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activated dissociation (CAD) isoften used to generate fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In some preferred embodiments, fatty acids in a sample are detectedand/or quantitated using MS as follows. The samples are first purifiedby liquid-liquid extraction. Then, the purified sample is subjected toliquid chromatography, preferably on an analytical column (such as aHPLC column) and the flow of eluted fatty acids from the chromatographiccolumn is directed to the ionization source of an MS analyzer. Fattyacids from the chromatographic column are ionized via APCI in negativeionization mode. The generated ions pass through the orifice of theinstrument and enter a series of three quadrupoles (Q1, Q2, and Q3). Q1acts as a mass filter, allowing selection of ions (i.e., selection of“precursor” ions) to pass into Q2 based on their mass to charge ratio(m/z). Q2 acts as a collision chamber where precursor ions arefragmented into fragment ions. Q3 acts as a mass filter allowing forselection of ions (i.e. fragment ions) based on their m/z. The threequadrupoles select for ions with the mass to charge ratios of fattyacids ions of interest. Ions with the correct mass/charge ratios areallowed to pass the quadrupoles and collide with the detector.

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theareas under the peaks corresponding to particular ions, or the amplitudeof such peaks, may be measured and correlated to the amount of theanalyte of interest. In certain embodiments, the area under the curves,or amplitude of the peaks, for VLCFA and/or BCFA ions are measured todetermine the amount of VLCFA and/or BCFA in the original sample. Asdescribed above, the relative abundance of a given ion may be convertedinto an absolute amount of the original analyte using calibrationstandard curves based on peaks of one or more ions of an internalmolecular standard.

The following Examples serve to illustrate the invention. These Examplesare in no way intended to limit the scope of the methods. In particular,the Examples demonstrate quantitation of very long chain fatty acids(VLCFA) and branched chain fatty acids (BCFA) by mass spectrometry, andthe use of VLCFA-²H₃ and BCFA-²H₃ as internal standards. The use ofVLCFA-²H₃ and BCFA-²H₃ as internal standards are not meant to belimiting in any way. Any appropriate chemical species, easily determinedby one in the art, may be used as an internal standard.

EXAMPLES Example 1: Hydrolysis of Fatty Acid Esters and Liquid-LiquidExtraction

The following hydrolysis and liquid-liquid extraction techniques wereconducted on controls, standards, and patient serum samples to preparesamples for mass spectrometric analysis. Plasma samples were also testedwith similar results (not shown).

First, 100 μL of an isotopically labeled VLCFA-²H₃ and/or BCFA-²H₃internal standard mixture was mixed with 200 μL aliquots of eachstandard, control, and patient sample. 200 μL of 1.0M NaOH were added tothe sample mixture and the NaOH-treated mixture was heated attemperatures between about 120° C. and 125° C. for about 60 minutes. Themixtures were then removed and allowed to cool for about 5 minutes.

After cooling, 400 μL of 5M HCl was added to each cooled sample mixtureand vortexed briefly. The sample mixture was then re-heated attemperatures between about 120° C. and 125° C. for about 75 minutes.After incubation was complete, the mixtures were again allowed to coolfor about 5 minutes.

3.5 mL of 10% ethyl acetate in hexane was then added to each sample; theresulting mixtures vortexed for 3 minutes and centrifuged at 2500 rpmfor 5 minutes. After centrifugation, the samples were placed in amethanol/dry ice bath for 5 minutes to freeze the aqueous later. Theorganic layer was then decanted off, dried to completion under a flowingnitrogen gas manifold, and reconstituted in 150 μL of ethanol.

The resulting samples were transferred to HPLC vials and placed in anautosampler for analysis.

Example 2: Purification of VLCFA and/or BCFA with Liquid Chromatography

Sample injection was performed with an Agilent Technologies G1367BAutosampler.

The autosampler system automatically injected an aliquot of the aboveprepared reconstituted samples into a Thermo Scientific BDS HYPERSIL™(C18 alkyl chain) HPLC column (3 μm particle size, 100×2.1 mm, fromThermo Scientific). An HPLC gradient was applied to the analyticalcolumn, to separate VLCFA and BCFA from other components in the sample.Mobile phase A was 20 mM ammonium acetate and mobile phase B was 82%acetonitrile in methanol. The HPLC gradient started with an 82% solventB which was ramped to 90% in approximately 1 minute, then ramped up to95% for another minute, and held at that percentage for approximately 36seconds, before being ramped back down to 90% over the next one minuteand 18 seconds, and then down to 82% over the next 24 seconds. Columnflow rate during solvent application was about 0.85 mL/min. Pristanicacid, phytanic acid, docosanoic acid, tetracosanoic acid, andhexacosanoic acid were observed to elute off the column at approximately1.43 minutes into the gradient profile.

Example 4: Detection and Quantitation of VLCFA and/or BCFA by MS

MS was performed on the above eluted samples using an Agilent 6130Single Quadrupole Mass Spectrometer. Liquid solvent/analyte exiting theanalytical column flowed to the ionization interface of the MS/MSanalyzer. The solvent/analyte mixture was converted to vapor in thetubing of the interface. Analytes in the nebulized solvent were ionizedby APCI.

Ions passed to the quadrupole mass selector (Q1), which selectedpristanic acid, phytanic acid, docosanoic acid, tetracosanoic acid, andhexacosanoic acid ions with mass-to-charge ratios (m/z) of 297.3±0.5,311.2±0.5, 339.3±0.5, 367.3±0.5, and 395.4±0.5, respectively. Theselected ions then traveled to a detector for counting. Massspectrometer settings used for this Example are shown in Table 1.Simultaneously, the same process using isotope dilution massspectrometry was carried out with internal standards: pristanicacid-²H₃, phytanic acid-²H₃, docosanoic acid-²H₃, tetracosanoicacid-²H₃, and hexacosanoic acid-²H₃. The masses monitored for detectionand quantitation during validation on negative polarity are shown inTable 2.

TABLE 1 Mass Spectrometer Settings for Detection of Very Long ChainFatty Acids and Internal Standards (Negative Ionization) MassSpectrometric Instrument Settings Gas Temperature 350° C. VaporizerTemperature 245° C. Drying Gas Flow 12.0 L/min Nebulizer Pressure 50psig Vcap (positive) 4000 V Vcap (negative) 1800 V Vcharge (positive)2000 V Vcharge (negative) 1000 V Corona (positive) 5.0 μA Corona(negative) 40 μA

TABLE 2 Mass-to-Charge ratios monitored for Very Long Chain Fatty Acidsand Internal Standards (Negative Ionization) Analyte Ion (m/z) Pristanicacid 297.3 ± 0.5 Phytanic acid 311.2 ± 0.5 Docosanoic acid 339.3 ± 0.5Tetracosanoic acid 367.3 ± 0.5 Hexadocosanoic acid 395.4 ± 0.5 Pristanicacid-²H₃ 300.3 ± 0.5 Phytanic acid-²H₃ 314.3 ± 0.5 Docosanoic acid-²H₃343.3 ± 0.5 Tetracosanoic acid-²H₃ 371.4 ± 0.5 Hexadocosanoic acid-²H₃399.4 ± 0.5

Exemplary chromatograms for pristanic acid, phytanic acid, docosanoicacid, tetracosanoic acid, and hexadocosanoic acid obtained from analysisof standard samples are shown in FIGS. 1A, 2A, 3A, 4A, and 5A,respectively. Exemplary chromatograms for pristanic acid-²H₃, phytanicacid-²H₃, docosanoic acid-²H₃, tetracosanoic acid-²H₃, andhexadocosanoic acid-²H₃ obtained from analysis of standard samples areshown in FIGS. 1B, 2B, 3B, 4B, and 5B, respectively.

An exemplary chromatogram obtained from a serum sample is shown in FIGS.6A, 7A, 8A, 9A, and 10A (each showing labeled peaks from pristanic acid,phytanic acid, docosanoic acid, tetracosanoic acid, and hexadocosanoicacid, respectively). Exemplary spectra obtained from mass spectrometricanalysis a serum sample as described above are shown in FIGS. 6B(pristanic acid), 7B (phytanic acid), 8B (docosanoic acid), 9B(tetracosanoic acid), and 10B (hexadocosanoic acid). The spectra werecollected by scanning Q1 across a m/z range of about 280-305 forpristanic acid, 295-341 for phytanic acid, 328-371 for docosanoic acid,360-385 for tetracosanoic acid, and 369-422 for hexadocosanoic acid.

Example 5: Linearity of Detection for VLCFA and BCFA

Calibration curves were prepared for the quantitation of pristanic acid,phytanic acid, docosanoic acid, tetracosanoic acid, and hexadocosanoicacid in serum by analysis of standards across a range of concentrations.Exemplary calibration curves for the determination of pristanic acid andphytanic acid in serum specimens are shown in FIGS. 11-12, respectively.Exemplary calibration curves for the determination of docosanoic acid,tetracosanoic acid, and hexadocosanoic acid in serum specimens are shownin FIGS. 13-15, respectively. Analysis of the data generated for thesestandards demonstrates that the assay exhibits linear response forpristanic acid in the concentration range of about 0.15-60 μmol/L; forphytanic acid in the concentration range of about 0.24-200 μmol/L; fordocosanoic acid in the concentration range of about 0.54-300 μmol/L; fortetracosanoic acid in the concentration range of about 0.36-300 μmol/L;for hexacosanoic acid in the concentration range of about 0.15-60μmol/L.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining an amount of oneor more underivatized fatty acids in a sample by mass spectrometry, themethod comprising: (a) subjecting the sample containing an amount of oneor more underivatized fatty acids to an ionization source to generateone or more underivatized fatty acid ions detectable by massspectrometry, wherein the one or more underivatized fatty acids isselected from the group consisting of docosanoic acid, tetracosanoicacid, hexacosanoic acid, pristanic acid, and phytanic acid; (b)determining an amount of the one or more underivatized fatty acid ionsby mass spectrometry; and (c) determining the amount of the one or moreunderivatized fatty acids in the sample from the amount of the one ormore underivatized fatty acid ions determined in step (b).
 2. The methodof claim 1, wherein said fatty acid is pristanic acid.
 3. The method ofclaim 1, wherein said fatty acid is phytanic acid.
 4. The method ofclaim 1, wherein said fatty acid is docosanoic acid.
 5. The method ofclaim 1, wherein said fatty acid is tetracosanoic acid.
 6. The method ofclaim 1, wherein said fatty acid is hexacosanoic acid.
 7. The method ofclaim 1, wherein the ionization source is electron ionization, chemicalionization, electrospray ionization (ESI), photon ionization,photoionization, atmospheric pressure photoionization (APPI), laserdiode thermal desorption (LDTD), fast atom bombardment (FAB), liquidsecondary ionization (LSI), matrix assisted laser desorption ionization(MALDI), field ionization, field desorption, thermospray ionization,plasmaspray ionization, surface enhanced laser desorption ionization(SELDI), inductively coupled plasma (ICP), or particle beam ionization.8. The method of claim 1, wherein the sample is subjected toliquid/liquid extraction prior to ionization.
 9. The method of claim 1,wherein the one or more fatty acids are subjected to a liquidchromatography column prior to ionization.
 10. The method of claim 9,wherein the liquid chromatography column comprises a high performanceliquid chromatography (HPLC), reverse phase liquid chromatography(RPLC), turbulent flow liquid chromatography (TFLC), or high turbulenceliquid chromatography (HTLC).
 11. The method of claim 1, wherein themethod further comprises determining an amount of one or more internalstandards added prior to step (a).
 12. The method of claim 11, whereinthe internal standard is pristanic acid-²H₃ phytanic acid-²H₃,docosanoic acid-²H₃, tetracosanoic acid-²H₃, or hexacosanoic acid-²H₃.13. A method of diagnosing or monitoring a peroxisomal disordercomprising determining an amount of one or more fatty acids in a patientsample by steps of claim
 1. 14. The method of claim 13, wherein anabnormal level of fatty acids is indicative of the peroxisomal disorder.15. The method of claim 14, wherein the peroxisomal disorder isZellweger syndrome, pseudo-Zellweger syndrome, infantile and adultRefsum disease, adrenoleukodystrophy, rhizomelic chondrodysplasiapunctata type 1 (RCDP-1), D-bifunctional protein deficiency, or acyl-coAoxidase deficiency.
 16. A method for determining an amount of one ormore underivatized fatty acids in a sample by mass spectrometry, themethod comprising: (a) subjecting the sample containing an amount ofunderivatized fatty acids to a hexane extraction, wherein the one ormore underivatized fatty acids is selected from the group consisting ofdocosanoic acid, tetracosanoic acid, hexacosanoic acid, pristanic acid,and phytanic acid; (b) subjecting the sample to liquid chromatography;(c) subjecting the sample to an ionization source to generate one ormore underivatized fatty acid ions detectable by mass spectrometry; (d)determining an amount of the one or more underivatized fatty acid ionsby mass spectrometry; and (e) determining the amount of theunderivatized fatty acids in the sample from the amount of the one ormore underivatized fatty acid ions determined in step (d).
 17. Themethod of claim 16, wherein said fatty acid is pristanic acid.
 18. Themethod of claim 16, wherein said fatty acid is phytanic acid.
 19. Themethod of claim 16, wherein said fatty acid is docosanoic acid.
 20. Themethod of claim 16, wherein said fatty acid is tetracosanoic acid. 21.The method of claim 16, wherein said fatty acid is hexacosanoic acid.22. The method of claim 16, wherein the ionization source is electronionization, chemical ionization, electrospray ionization (ESI), photonionization, photoionization, atmospheric pressure photoionization(APPI), laser diode thermal desorption (LDTD), fast atom bombardment(FAB), liquid secondary ionization (LSI), matrix assisted laserdesorption ionization (MALDI), field ionization, field desorption,thermospray ionization, plasmaspray ionization, surface enhanced laserdesorption ionization (SELDI), inductively coupled plasma (ICP), orparticle beam ionization.
 23. The method of claim 16, wherein saidsample is subjected to an acid hydrolysis prior to ionization.
 24. Themethod of claim 16, wherein the method further comprises determining anamount of one or more internal standards added prior to step (a). 25.The method of claim 24, wherein the internal standard is pristanicacid-²H₃ phytanic acid-²H₃, docosanoic acid-²H₃, tetracosanoic acid-²H₃,or hexacosanoic acid-²H₃.
 26. A method of diagnosing or monitoring aperoxisomal disorder comprising determining an amount of one or morefatty acids in a patient sample by steps of claim
 16. 27. The method ofclaim 26, wherein an abnormal level of fatty acids is indicative of theperoxisomal disorder.
 28. The method of claim 27, wherein theperoxisomal disorder is Zellweger syndrome, pseudo-Zellweger syndrome,infantile and adult Refsum disease, adrenoleukodystrophy, rhizomelicchondrodysplasia punctata type 1 (RCDP-1), D-bifunctional proteindeficiency, or acyl-coA oxidase deficiency.